Cancer-targeting antibodies can provide a versatile starting point for generation of tumor-specific imaging agents. In particular, through protein engineering, recombinant antibody fragments can be produced that are optimized for in vivo tumor targeting, blood clearance, and normal tissue distribution and clearance patterns. Following conjugation and radiolabeling of such fragments with positron-emitting radionuclides such as Cu-64 and 1-124, engineered antibody fragments can be used for sensitive, high-resolution imaging in murine tumor xenograft models using small-animal imaging modalities such as microPET (positron emission tomography). We propose to develop high-avidity multimeric cancer imaging agents utlizing a novel protein multimerization technology. As a model system, single chain antibodies specific for carcinoembryonic antigen (CEA) will be reformatted to generate dimers and tetramers. 1) Following bacterial expression and purification biochemical and binding properties will be determined in vitro. 2) Recombinant antibodies will be labeled with radioiodine or conjugated with a chelating moiety (DOTA) for radiometal labeling for binding and biodistribution studies. Proteins will also be radiolabeled with 1-124 and Cu-64 for microPET imaging studies. In addition, a set of high-avidity multimeric anti-CEA antibody fragments modified with C-terminal Cys residues will be developed for site-specific conjugation and radiolabeling. 3) Performance of the antibody agents will be evaluated in athymic mice bearing CEA-positive (LS1741) and CEA-negative (C6) xenografts, in biodistribution studies and by serial microPET imaging studies in living mice. 4) The tumor targeting biodistribution, and imaging potential of the novel antibody constructs will be evaluated. The dimer and tetramer will be compared to determine whether the high avidity imparted by four binding domains enhances overall in vivo performance. The novel fragments will also be evaluated against existing antibody formats. Knowledge gained through this work will accelerate the development of cancer-specific imaging agents derived from any of a variety of antibodies specific for tumor-associated antigens, for single-photon or PET imaging applications.